The recommended dietary allowance for vitamin C (ascorbate) is based on preventing the deficiency disease scurvy. Optimal vitamin C requirements are unknown. We proposed that optimal requirements can be determined using the concept of in situ kinetics. In situ kinetics has biochemical and clinical components. The goal of the biochemical component is to determine vitamin C molecular function in relation to its concentration. For these studies vitamin C function is investigated in human fibroblasts, lymphocytes, and neutrophils. To determine how intracellular concentration is regulated, vitamin transport has been characterized. Ascorbate transport is concentration dependent, saturable, mediated by a sodium dependent carrier, and inhibited by newly synthesized vitamin C analogs. Oxidized ascorbate, dehydroascorbic acid is transported separately by GLUT I and immediately reduced intracellularly to ascorbate. Two distinct proteins have been isolated which mediate intracellular reduction in neutrophils. Cloning of these proteins is underway. These studies indicate that vitamin C function in neutrophils may be to protect neutrophils from their own oxidants. One function of vitamin C in lymphocytes may be regulation of regulation of gene transcription. The overall findings show that kinetics for vitamin C function in situ can be determined in relationship to vitamin concentration in situ. The clinical component of in situ kinetics is to determine how vitamin concentrations are achieved in normal humans as a function of dose and whether concentrations human achieve are in the range required for molecular functions of the vitamin. A clinical trial has been completed in normal men which investigated the relationship between vitamin C dose and its concentration in plasma and tissues, bioavailability, urinary excretion, and potential adverse effects. A clinical trial in women is underway. Based on biochemical and clinical in situ kinetics, we can recommend for the first time an optimal vitamin C requirement in health humans.